Adenylyl cyclase is a family of enzymes that catalyze the formation of adenosine 3':5'-monophosphate (cAMP) from ATP. It also belongs to a superfamily of membrane-bound transporter and channel proteins. Mammalian forms of the enzyme are stimulated or inhibited by hormones via G-protein- linked cell-surface receptors. Adenylyl cyclases are also inhibited via a cytoplasmic domain ("P"-site) by adenine nucleoside 3'-phosphates that derive from nucleic acid metabolism. Thr proposed investigations focus on this inhibition of adenylyl cyclases via the "P"-site. The several isozymes of adenylyl cyclase, whether extracted from various tissues or expressed in Sf9 cells, exhibit different sensitivities to "P"-site- mediated inhibition that may reflect differing physiological roles for this inhibition in tissues expressing the respective forms of the enzyme. The general focus of the proposal is to utilize biochemical probes to define structural characteristics of the inhibitory domain and to utilize selected probes to define its locus within selected expressed wild type and mutated isozymes. A very important corollary to this approach is to identify and quantify naturally occurring "P"=site inhibitors and to identify the pathways of their synthesis and degradation. These approaches build on our recent discovery that 3'-polyphosphorylated adenine nucleotides are a very potent, previously undescribed class of inhibitor of adenylyl cyclases. Characteristics of "P"-site-mediated inhibition will be determined for the different adenylyl cyclases. Characteristics of "P"-site-mediated inhibition will be determined for the different adenylyl cyclase isozymes, with emphasis on ligand specificity and the role of post-translational modification through phosphorylation-dephosphorylation mechanisms, specifically involving protein kinases C and A. Recently synthesized ligands will be tested further and additional site-selective ligands will be made. Emphasis will be on selective and high-affinity probes that can be used for covalent attachment tot he "P"-site, that can be labeled with radioactive tracers and/or with fluorescent indicators, that are metabolically and chemically stable, and probes that can be used either directly or as prodrugs to inactivate adenylyl cyclase in intact cells. Radioactive covalent affinity probes will be used to tag the "P"-site. Initial probes will be based on 3'-polyphosphorylated adenine nucleosides. Labeled peptides will be isolated and sequenced an these will be compared with those of the adenylyl cyclases and other proteins. Cellular levels of "P"-site ligands will be determined. High affinity ligands will be used for the identification and quantification of naturally occurring "P"-site ligands and of possibly other proteins to which this class of nucleotide binds. The proposed studies will provide a unique handle on adenylyl cyclase. Characteristics for this new class of modulator of adenylyl cyclases will be defined, and the regulatory links between this family of enzymes and nucleic acid metabolism will be strengthened.